This project concerns the functional roles of neurocan and phosphacan, two major nervous tissue-specific chondroitin sulfate proteoglycans whose biochemical properties and primary structures have previously been described by this laboratory. Despite their dissimilar structures, both proteoglycans bind specifically and with high affinity to neural cell adhesion molecules and the extracellular matrix protein tenascin. It is now planned to explore the roles of neurocan and phosphacan in neurobiological processes using both genetic approaches and studies on cultured cells. Utilizing a promoter that has been found to consistently drive the expression of a bacterial lacZ reporter gene in the limbic system of brain, establishment of new transgenic mouse lines will allow assessment of the effects of individually overexpressing in limbic structures the extracellular proteoglycan phosphacan or the full-length receptor-type protein tyrosine phosphatase (RPTP-zeta/beta), of which phosphacan is an alternative splicing variant. Another project concerns the potential roles of neurocan and phosphacan in the behavior of neuronal growth cones, which are the agents of axonal pathfinding and are responsible for the establishment of neuronal connections with target cells. Based on the results of our previous studies demonstrating potent inhibitory effects of neurocan and phosphacan on neuronal adhesivity and neurite outgrowth, it is now planned to examine their effects on nerve growth cone morphology, to determine whether contact with neurocan or phosphacan results in growth cone collapse, and whether the effects of proteoglycans on neurite outgrowth or growth cone avoidance are mediated by changes in cytoplasmic calcium concentration. Neurocan belongs to a family of structurally related proteoglycans which includes at least three additional members (aggrecan, versican, and brevican) all of which are also expressed to varying extents in nervous tissue. Gene targeting will be used to obtain information on the roles of proteoglycans in central nervous system function by examining the phenotype of mice in which the genes for neurocan and/or brevican have been deleted, as well as of mice lacking either neurocan or brevican and tenascin. In view of recent studies which demonstrated that the targeted deletion of genes for certain presumably "critical" proteins (such as tenascin) resulted in no phenotype, it has become apparent that structurally related or interacting proteins are in some cases able to compensate for the missing product of the deleted gene. Because neurocan (which interacts with tenascin) and brevican are likely to fit this pattern, it is important to examine the effects of deleting the genes for two, and possibly even three, of these proteoglycans and/or their binding partners that we have previously identified. It can be expected that our studies will significantly advance understanding of the roles of these extracellular matrix components not only in developmental processes but also in nervous tissue injury and repair (e.g.,following spinal cord injury or stroke), where there is experimental evidence that they may have therapeutic utility.